Decoding Viral Dark Matter: Metagenomic Prokaryotic Virus Characterization With Pharokka, Phold, and Phynteny

Viral metagenomics is an increasingly powerful tool for understanding the function and structure of viruses across the diverse environments of our planet. However, decoding the functional potential of prokaryotic viral metagenomes is extremely challenging. Pharokka, Phold, and Phynteny are complementary open-source prokaryotic viral genome annotation tools that utilize a variety of bioinformatics approaches to maximally annotate viral metagenomes. This article describes a protocol for installing and running these tools on a viral metagenomic dataset, followed by visualization of annotations using our client-side Phold Plot web assembly application. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Prokaryotic viral metagenome annotation with Pharokka Basic Protocol 2: Enhanced prokaryotic viral metagenome protein annotation using protein structures with Phold Basic Protocol 3: Further prokaryotic viral metagenome protein annotation using genome synteny and protein language models with Phynteny Basic Protocol 4: Visualization of prokaryotic viral metagenome annotations with Phold Plot web assembly application.